| 摘要: | 本研究主要是尋找一些參與人類精子發育過程中的基因。藉由cDNA微晶片分析，比較造精弁鉒妘揪爾A丸組織（MA和SCOS）與正常睪丸組織（NR）mRNA的表現差異，找出表現量具有明顯差異的特定基因，以real-time RT-PCR確認特定基因mRNA於造精弁鉒妘晴A丸組織中（NR, HS, MA, SCOS）表現結果與微晶片分析結果大部分相符合。針對BC009436 EST進一步分析基因特性及轉錄調節機制。NCBI比對人類BC009436不同物種的同源性基因為小鼠AK084948和大白鼠XM-222072，以Rapid amplification cDNA end反應，獲得人類BC009436、小鼠AK084948和大白鼠XM-222072完整cDNA序列分別由2185、2177及2115個核甘酸所組成。生物資訊工具預測：人類BC009436的基因位於其7號染色體上；小鼠AK084948的基因位於其5號染色體上；大白鼠XM-222072的基因位於其12號染色體上，皆由15個表現子所組成，基因體結構組成符合GT-AG規則。開啟讀碼框預測起始密碼（ATG）及終止密碼（TAG），經轉譯出的胺基酸序列於人類BC009436、小鼠AK084948和大白鼠XM-222072分別由647、648及648個胺基酸所組成。GCG軟體比對三種不同物種間核甘酸及胺基酸序列的相似度及相同性：人類BC009436與小鼠AK084948核甘酸比對結果其相似度及相同性皆為78.0%，胺基酸比對結果相似度及相同性分別為81.4%、78.3%；人類BC009436與大白鼠XM-222072核甘酸比對結果其相似度及相同性皆為78.8%，胺基酸比對結果相似度及相同性分別為80.9%、78.5%。蛋白質二級結構預測具有LRR及WD domain。RT-PCR顯示：人類BC009436和小鼠AK084948 mRNA主要大量表現於睪丸組織；小鼠AK084948 mRNA主要表現在精子形成過程的晚期。探討人類BC009436基因的轉錄調節機制，以5’-RACE-PCR獲得轉錄起始點（+1），於轉錄起始點附近約1.9 kb片段預測啟動子區域位於-198至+53間，為GC-rich序列，缺乏TATA box（TATA-less promoter）和CCAAT box，具有NF-kB、AP-2、GCF、T-Ag和Sp1等轉錄因子結合位。人類BC009436轉錄起始點附近約1.3 kb（-1270/+53）片段由5’端進行序列刪除，分析不同片段（-1270/+53, -1270/-1, -827/+53, -827/-1, -515/+53, -515/-1, -198/+53, -198/-1及-198/+100）於不同細胞株中啟動子活性表現情形。顯示293T細胞（人類腎臟細胞株）、TM3細胞（mouse Leydig cell ）、TM4細胞（mouse Sertoli cell）和GC-2spd(ts)細胞（mouse spermatocyte）分析結果定義-198至+53片段為core promoter region。-198至+100片段活性表現於293T細胞, TM3細胞與TM4細胞的分析結果推測+53至+100間可能有positive regulator element存在；GC-2spd(ts)細胞中-198至+100片段活性分析結果推測+53至+100可能有negative regulator element存在。提出人類BC009436基因在germ cells（GC-2spd(ts)）與somatic cells（293T、TM3、TM4）有不同的轉錄調控方式。
The study is focused on the novel genes analysis in spermatogenesis of human. The gene expression pattern in testicular biopsies of men with sperma- togenic defect is analyzed by cDNA microarrays. The mRNA expression of BC009436 novel gene is significant different between SCOS, MA and NR. The microarray data is confirmed by real-time RT-PCR. The other mammalian species of mouse AK084948 and rat XM-222072 is isolated and characterized by BLAST. The full length cDNA of different speciesis obtained by Rapid amplification cDNA end methods. The full lengths of human, mouse and rat cDNA are 2185, 2177 and 2115 bp respectively. Human BC009436 gene conta- ined 15 exons and was mapped to chromosome 7. The homologous genes of mouse AK084948 and rat XM-222072 genes were mapped to chromosome 5 and chromosome 12 respectively. The ORF encode 647, 648 and 648 amino acids of human, mouse and rat respectively. Predict the protein containing leucine-rich repeats and WD domain. A search in the GCG web showed that the nucleotide sequence of human BC009436 was 78.0% similarity and identity, amino acid sequence was 81.4% similarity and 78.3% identity to that of mouse AK084948; the nucleotide sequence of human BC009436 was 78.8% similarity and identity, amino acid sequence was 80.9% similarity and 78.8% identity to that of rat XM-222072. The mRNA expressed highly in testis by RT-PCR in human and mouse multiple tissues assay. The mouse AK084948 mRNA is stage-specific expression from the elongated spermatids to spermatozoa during mouse testi- cular development. Human BC009436 gene may involve in the regulation of late spermatogenesis development. To study the mechanisms of human BC009436 transcriptional regulation, we used 5’-RACE to identify transcription start site. The prediction promoter region of human BC009436 gene in -198 to +53 that the GC-rich region; lacks a TATA box (TATA-less promoter) and CCAAT box, containing putative transcription factor binding site of NF-kB, AP-2, GCF, T-Ag and Sp1. Luciferase reporter analysis of a 1.3 kb 5’-flanking sequence (-1270 to +53 with respect to the transcription start site). Serial deletions analysis promoter activity in 293T (Human embryonic kidney cell lines), TM3 (mouse Leydig cell), TM4 (mouse Sertoli cell) and GC-2spd(ts) (mouse spermatocyte) that revealed -198 to +53 fragment is core promoter region. The -198 to +100 fragment has strong promoter activity in 293T, TM3 and TM4, suggesting the +53 to +100 has presence of positive regulator element. GC-2spd(ts) cell that revealed +53 to +100 has presence of negative regulator element. |
